Vitamin

Jones, Burt P Koonsvitsky, Mary L Ebert, Lin, Birgit H Will, and John W Suttie The

potential

ABSTRACT tamin K status

was

living

Functional

subjects.

(E) assay]

assessed

concentrations

affected

by olestra.

Initial

olestra and placebo of 0.92 for normal

for 20 g olestra/d

in a 6-wk

study

different

between

intake

were

0.80

and

were 0.79

un-

for the

should

cating

20 g olestra/d

been

diaries subjects

was

low,

reflected

in those

diet

did

‘-‘#{243}O zg/

affect

mdi-

vitamin

sucrose

polyester,

vitamin

and

primary

dietary

is absorbed

source

by being

of vitamin

solubilized

K, is

nondigestible lipid in the intestinal with the absorption of fat-soluble

substances

of interference

(2-5).

lipophilicity

The

,

Olestra by gastric using

with

of the

usual

formed

name

from

solute

primarily

and

the

molecule.

the potential for 20 ingested chronically.

for the

mixture

the reaction

of hexa-

fat substitute is not absorbed

prothrombin

of long-chain

of the

ylated forms the impairment

design

shown

than

time

(PT)

that

complete

is not hydrolyzed

(7). Previous studies, as a measure, showed that

or partially

of vitamin K-dependent of vitamin K action

des-gamma

measurements

of vitamin

does

to reflect

changes

concentration

of

was to determine

affected

vitamin

if the ingestion

K status

concentration,

as measured

PT,

and

partial

(PTT).

and procedures was a double-blind, oftwo

health by laboratory

placebo-controlled,

groups

medical history, data. Vegetarians

ofnormal,

healthy,

carbox-

proteins resulting from (1 1, 12). These species can

parallel

de-

free-living aged

sub18-55

y

to be in good

physical examination, and and subjects on weight-loss

clinical diets

who might be consuming large amounts ofleafy vegetables, and people taking oral anticoagulants, were excluded. The study was approved by the study-site institutional review board (Pharmacobogy Research and Clinical Studies Institute, Fargo, ND). Signed informed consent was obtained from each subject. The subjects were randomly assigned within strata ofage, sex, and body mass index to yield two balanced groups. The groups consumed 20 g/d ofolestra or placebo (lard) in six cookies, eaten two

at each

meal,

chronic olestra

for 6 wk.

this amount

by the subjects

olestra does not affect vitamin K status (8-10). However, mdicators ofvitamin K status more sensitive than clotting times are now available. Recently developed methods allow the direct measurement

two adequacy

and methods

humans,

sucrose.

is a zero-calorie lipases (6) and

clinical

kinetics

and

and octaesters

acids

depends

in the intestine

study was to determine vitamin K status when

is the common

hepta-,

fatty

lipid

partitioning

The purpose ofthis g olestra/d to affect Olestra

degree

of nondigestible and

Subjects

sign consisting

in luminal

micelles (1). The presence ofa tract can potentially interfere on the amount

rapidly

phylloquinone

time

ofthe

of the

S:E was

of this study

thromboplastin

ratio

jects. One-hundred nine females and 1 12 males were enrolled in the study. They were determined

the

lipophilic

The

Echis of pro-

from

amount

(13).

in the diet

serum

The study

K, phyl-

bioavailability

Phylloquinone,

S:E,

Study

Introduction highly

objective

protease

of the total

K

199 1;53:943-6.

Olestra,

by

more

in blood

g olestra/d

venom

measure

synthesis.

K intake

phylloquinone

snake

(13).

a direct factor

in vitamin

the

quantification

in a bio-

measures,

not

K for clotting

of2O

that the

K status were unaffected decrease in phylloquinone

in the

Am J C/in Nutr

indicated

is therefore

The

serum concentrations, were not significantly

food

ofthe

availability

KEY WORDS

in the plasma

(S:E)

times

with

(E) allows

thrombin

Weekly

have

activation

carinatus

clotting

S:E values

groups.

whereas

[Simplastin#{174} (5)-Ecarin#{174}

classical

measures of vitamin where any significant

loquinone,

vifree-

olestra13

B Jones,

and

d. Sensitive population

status.

202

groups, respectively, compared with a value reference plasma. At week 6 the value was

phylloquinone

that

Michaelle

to affect

involving

consuming

prothrombin

0.81 for both groups. Mean phylloquinone expressed as differences from baseline, average

subjects

(14).

Twenty

intake of olestra was divided among

is unlikely

to be eaten

a food for delivering

On

the

of olestra

basis

of previous

studies

in

is expected

grams

to be well tolerated per day exceeds the estimated

under expected-use conditions. The all three meals because 20 g olestra

at a single

the olestra

meal.

because

Cookies

were

they represent

chosen

as

a realistic

I From the Procter & Gamble Company, Cincinnati, OH, Department of Biochemistry, University of Wisconsin-Madison. 2 Supported by the Procter & Gamble Company, Cincinnati,

and

the

OH.

thromboplastin, eg, Simplastin (5; Warner Lambert, Morris Plains, NJ) allows quantification of the amount of carboxylated, biologically active prothrombin

reprint requests to KD Lawson, The Procter & Gamble Company, Winton Hill Technical Center, 6071 Center Hill Road, Cmcinnati, OH 45224. Received June 25, 1990. Accepted for publication October 17, 1990.

Am J C/in Nuir

for Ginical

also

plasma

be

assessed

by

functional

clotting

assays.

Activation

of

by a commercial

1991;S3:943-6.

Printed

in USA.

© 1991 American

Society

3

Address

Nutrition

943

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D Yvonne Peter YT

K status of free-living

944

JONES

TABLE

ET

AL

1

Effect ofolestra

or placebo

ingestion

on phylboquinone

Group

nutriture4

Week 0

Week

2

Week

Week 6

4

S:E Olestra Placebo

Prothrombin

0.02 ± 0.02

0.78 0.79

0.44 0.53

±

± 0.02 ± 0.01

0.79 0.81

±

0.04 0.04

0.02 ± 0.01

0.33

± 0.02

0.37

±

0.55

± 0.04

0.55

±

0.8 1 ± 0.02 0.81 ± 0.01

(nmol/L)

time

±

0.04 0.07

0.40 0.48

0.04 ± 0.04 ±

(s)

Olestra Placebo Partial

±

12.2 12.2

thromboplastin

0.04 0.03

ND ND

ND ND

12.1 12.2

± 0.03

±

0.5

ND

ND

27.6

± 0.3

ND

ND

28.3

±0.3

±

±

0.03

time (s)

Olestra

30.7

±

Placebo

29.7

±0.5

4 1 ± SEM; n = 82 for olestra and n = 88 for placebo. S:E, ratio ofSimplastin#{174} to Ecarin#{174};ND, not determined. Repeated-measures analysis of variance (RMA) indicated no statistically significant differences between groups for S:E (P 0.87) or serum phylboquinone values expressed as differences from baseline (P = 0.22). RMA of absolute serum phylboquinone concentrations indicated a significant olestra effect (P = 0.002). Prothrombin time and partial thromboplastin time were not significantly different between groups at the P 0.05 level (Wilcoxon rank-sum test).

use of this material actual consumption.

and

provide

a simple

means

of calculating

samples were collected by venipuncture for and serum phylloquinone measurements at baseline and at weeks 2, 4, and 6. On days ofbbood collection, subjects Fasting

S:E,

blood

PT, PTf,

consumed

the

test

cookies

after

blood

was

taken.

and plasma were immediately frozen by immersion in ice-acetone bath and stored at -20 #{176}C until analyzed.

Serum

a dry Serum

phylboquinone

was

assayed

by using

methodology

based

on that

reported by Haroon et al (1 5). S:E measurement was based on procedures previously described (13, 16). Gotting times were determined by an accredited clinical laboratory (North Central Laboratories, Inc, Fargo, ND). The subjects were provided with weekly diaries and with prelisted

common

foods

(eg, green

vegetables,

eggs,

and cereals known to contain phylloquinone) to record their daily intake of those foods. phylboquinone

consumed

was

estimated

by using

standard

from

the

The

milk

and were asked daily intake of

amounts

food-composition

products,

of each

tables

food

(17).

Materials

Olestra was prepared from fatty acids from soybean oil and sucrose (18) and consisted of > 99% octa- and heptaesters. The composition ofthe fatty acids in the ester groups was C16, 10%; C18, 54%; C14:1 (cis), 20%; C18:2, 14%; others, < 2%. Vitamin Kfree triglyceride was used

OH)

Statistical

(lard

Cincinnati

Bakers

the placebo

Supply,

Cincinnati,

cookies.

analyses

phylloquinone

(RMA) serum performed

boquinone

to compare

oftreatment

and time

concentrations,

phylboquinone on differences

rank-sum groups

To account concentrations, from baseline

Analysis

test were with

on S:E and on

repeated-measures

was performed.

concentrations.

the Wilcoxon

Results Ninety-nine

subjects

in the olestra

completed

group

the study.

and

103 subjects

the placebo

group

Eleven

subjects

olestra group like cookies,

withdrew for the following reasons: two had job transfers, one had elective

in

in the

three did not surgery, one

had gastrointestinal distress, and four gave no reason. One olestra subject was disqualified for noncompliance. Seven placebo subjects did not complete the study: three gave no reason, two were disqualified

for noncompliance,

one

developed

mononucleosis,

and one developed gastroenteritis. The average amount of olestra consumed by the subjects completing the study was 19.7 g/d. Over 75% ofthe subjects reported that they ate all ofthe cookies. Only subjects who had consumed > 90% of the cookies and had blood samples collected and analyzed at all four time points were included in the statistical analyses. Eighty-two subjects in the olestra group and 88 in the placebo group met these criteria. Results of the analyses are shown in Table 1. Initial S:E values were 0.80 and 0.79 for the olestra and placebo groups,

respectively,

and

were

essentially

unchanged

during

the

study. The S:E values obtained from a normal reference plasma during the course ofthe study, with the reagents used to analyze the test samples, ranged from 0.87 to 0.99 (0.92 ± 0.03, ± SD). RMA of the S:E values showed no statistically significant differences between the groups (P 0.87). PT and PTT did not differ between groups at either measurement. There were no =

the effect serum

of variance in baseline yses were

from

to prepare

To determine

point

performed at each time point. SAS computer software (19) was used for all statistical calculations. All comparisons were made at the two-sided 0.05 significance level.

of variance

also performed

respect

analysis

for differences the same ofserum (ANOVA)

at each

to phylboquinone

analphyland

time serum

concentration and S:E. To test for differences in PT and PTT between groups, a t test and the Wilcoxon rank-sum test were

statistically RMA

significant of absolute

changes serum

in these

indices

phylloquinone effect (P = 0.002).

cated a significant olestra serum phylboquinone concentrations

with

time.

concentrations However,

ofthe

two groups

by 1 7% at baseline, therefore comparison baseline is more appropriate than comparison

of differences ofabsolute

mdithe mean

differed from values

for assessing potential treatment effects. The differences from were not significantly different (P 0.22) between the groups. Analysis of the data in terms of two subgroups, those baseline

=

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Serum phylloquinone Olestra Placebo

0.80 0.79

OLESTRA with

initial

plasma

(0. 18 nmol/L), baseline

phylloquinone

produced

did

not

differ

above

the

same

or below

results.

significantly

AND the

placebo

from

for either

and

significance

groups

level,

in

trends

over

time

between

the

two

ofthe

food

diaries

indicated

that

ate an average of 57 zg phylloquinone/d group ate 64 &g/d. The difference between not statistically significant by RMA.

the olestra

quinone this

would

study

serum

considered

to be the

primary

have

had

indicator

of vitamin

K

been

they

hospital

value

et al (13)

was

found

diet.

that

in almost

a 50%

centrations,

from

21 was

after

9 d on the

d on

a restricted

reduced

decrease

ofthe

restricted values

this

were

range

sufficiency,

S:E

of the

laboratory

subjects

a condition

may where

reference have

any

plasma

been

at the

interference

was

0.92

borderline with

Mean

serum

within

spection

K

that

the

ofthe

they

normal food

were

of vitamin

intakes

of -60

L).

the borderline season (late

of the

restricted

diet

with

50 ig

phyllo-

only

increased

serum

phylloquinone

concentrations

from

0.95

to 1.25 nmol/L, a value significantly lower than the prerestricted value. PT values were not different between prerestricted and restricted periods. Measurement of urinary ‘y-carboxyglutamic acid

(Gla)excretion

intake urinary

confirmed the effect ofdietary phylloquinone assay. The results suggest that S:E and

on the S-E excretion rate are both

loquinone

concentration

more

sensitive

is to changes

than

serum

in phylloquinone

Gla phyl-

intake.

by Suttie No

et al (1 3) in their

and

from

0.44

to 2.4 nmol/L

in subjects

for whom

the

of the study. absorption

or

is greatly

Had under

two groups,

comparison

ofdifferences

from

baseline

the

is the most

during

which

resulted con-

with

decrease 500

increased

than

g/d

for

the serum

prerestricted

con-

in our study

pop-

nmol/L in this

amounts

than

the 80-tg/d

utilization

in excess

principal

mean

intake

of college

olestra these

students. moderate

of dietary

vitamin

sensitive

measure

in a subject

ofvitamin

The

and the was on

because of the (North Dakota)

affected

phylloquinone

of marginal

population

K status.

The

initial

vitamin

whose

that any significant have been reflected

decrease in phylloquinone by a change in status.

K

requirement.

possibly location

significantly conditions

daily

reported

detecting

of the

In-

showed

of the The

of

(20).

study

phylloquinone

the effect would have been detected. ingestion of 20 g olestra/d for 6 wk did not affect study

S:E, a

was conducted

K status

was

such

absorption should The power of the test

was such that a 10% difference in S:E between have been detected. Olestra also did not produce

groups would any significant

concentrations when the difconcentrations of the two Twenty g olestra/d cor29% ofthe mean total dietary

fat intake of average US adult males and females, respectively and represents a high chronic intake. The results of this study indicate that the chronic ingestion

assistance

between

diet

vegetables.

of vitamin K sufficiency, winter to early spring) and

phylloquinone

considerably

of the Suttie

the serum phylloquinone concentrations, data all suggest that our subject population

food-diary

of 20 g olestra/d

differed

30% diet.

phylloquinone

and

is capable

absorption

intake

in the normal range. The cloning times and the serum phylloquinone concentration data shown in Table 1 support the conclusion that olestra had no significant effect on vitamin K status. Because baseline serum concentrations

-

a significant

leafy population

methodology

in the

S:E values,

intake, The

liberal

K, green

;zg are lower

current

changes when

consuming

(2 1),

diet

of

restricted

subjects

ranged having

S:E remained

value

of 0.29-2.64

differences in serum phylloquinone ference between the initial serum groups were taken into consideration. responds to substitution of 19% and

stricted

an

of 1 .0 1 nmol/L, measured in a large population, but

ofthe

Other evidence ofthe lack ofcorrelation between absolute serum phylloquinone concentrations and vitamin K status, as measured by the S-E assay, is found in the work ofAllison Ct al(16). These researchers found that serum phylloquinone concentrations

from < 0.22 to 2.2 nmol/L in healthy adult male subjects subnormal S:E values after 9 d of a phylloquinone-re-

in 3 d after

concentrations

diaries

not

sources

Supplementation

by that

was substituted

77 to 37 g/d,

to greater

phylloquinone

intake or utilization by olestra would have been easily detected. The size of the test would have allowed a change of 0.08 in S:E to be detected with 80% power. The sensitivity of S:E to changes in vitamin K intake is illustrated by the work ofSuttie et al (13). These authors report that subjects who consumed a vitamin K-deficient diet for 3 wk showed a significant decrease in S:E (from 1.02 to 0.91) and in plasma phylboquinone concentrations (from 2.02 to 0.95 nmol/ quinone/d for 12 d, which raised the average phylloquinone intake to above the prerestricted amount, restored S:E values to 0.98 (not significantly different from prerestricted amounts) but

covered

centrations.

of

vitamin

concenphylbo-

observed

50%

and

S:E to prerestricted

the mean technique

the

(0.05

igJd)

serum

nmol/L,

concentrations

time

diet,

from

in mean

1.91 to 1.01

Supplementation

12 d returned

(2-5

A steady-state

reached

intake

the

fell

diet

ulation were lower than with the same analytical

the

group

et al (16)

concentrations

phylboquinone-free

starting

.

thus

for this

phylboquinone over the 6 wk

placebo

within

Allison

phylloquinone

study

RMA

groups

serum phylloquinone absorption of dietary

status in this study. At the start of the study, the mean S:E for the olestra group was 0.80 and for the placebo group was 0.79. At the end of6 wk, both groups had a mean S:E of0.8 1 Therefore, olestra did not affect vitamin K status as reflected by S:E. The normal S:E value depends upon the specific reagents. In ± 0.03,

in the

detected

occurred.

phylboquinone

in S:E. S:E was

effects.

the two

in mean serum (0.04 nmol/L)

group change

Significant changes in S:E and trations resulting from decreased

phylloquinone

Discussion

treatment

between

nmol/L).

for a normal

group

whereas the placebo the two groups was

potential

The net change

0.22).

=

for

difference

concentration for the olestra was not different from the

essentially

for any index.

Inspection

(P

index

olestra

The

to look

no significant

population We thank Pharmacology

will

not

affect

under

expected

James

D Carlson,

Research in conducting phylboquinone analyses.

vitamin

usage Albert

K status

of the

conditions. Dicta, Jr, and Karen

general

B Stein of

and Clinical Studies Institute, Fargo, ND, for this study and Melissa Kling for conducting the

Downloaded from https://academic.oup.com/ajcn/article-abstract/53/4/943/4715070 by McMaster University Library user on 03 February 2019

0.05

subgroup.

way

showed

P values were 0.22 and 0.06 for the above-median and below-median subgroups, respectively. ANOVA indicated that the absolute serum phylloquinone concentrations were statistically significantly different (P 0.05) between the two groups at weeks 2 and 4 but not at week 6. The subjects tended to be consistent in all indices across the study. There was no statistically significant difference, at the two-sided

groups

945

K STATUS

meaningful

median

Differences

between

VITAMIN

946

JONES

References

and response

AC. Production of “prothrombin defito vitamins A, D and K. Proc Soc Exp Biol Ivy

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JT, Amelotti

JM,

Doisy

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AL 12. Shah DV, Swanson vitamin K-deficient

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4. Elliott

ET